1. Field of the Invention
The present invention relates to novel 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs, and the use of such compounds as pharmaceuticals.
2. Background Art
Retroviruses are small, single-stranded positive-sense RNA viruses. A retroviral particle comprises two identical single-stranded positive sense RNA molecules. Their genome contains, among other things, the sequence of the RNA-dependent DNA polymerase, also known as reverse transcriptase. Many molecules of reverse transcriptase are found in close association with the genomic RNA in the mature viral particles. Upon entering a cell, this reverse transcriptase produces a double-stranded DNA copy of the viral genome, which is then inserted into the chromatin of a host cell. Once inserted, the viral sequence is called a provirus. Retroviral integration is directly dependent upon viral proteins. Linear viral DNA termini (the LTRs) are the immediate precursors to the integrated proviral DNA. There is a characteristic duplication of short stretches of the host""s DNA at the site of integration.
Progeny viral genomes and mRNAs are transcribed from the inserted proviral sequence by host cell RNA polymerase in response to transcriptional, regulatory signals in the terminal regions of the proviral sequence, the long terminal repeats, or LTRs. The host cell""s protein production machinery is used to produce viral proteins, many of which are inactive until processed by virally encoded proteases. Typically, progeny viral particles bud from the cell surface in a non-lytic manner. Retroviral infection does not necessarily interfere with the normal life cycle of an infected cell or organism. However, neither is it always benign with respect to the host organism. While most classes of DNA viruses can be implicated in tumorigenesis, retroviruses are the only taxonomic group of RNA viruses that are oncogenic. Various retroviruses, such as the Human Immunodeficiency Virus (HIV), which is the etiological agent responsible for acquired immune deficiency syndrome (AIDS) in humans, are also responsible for several very unusual diseases of the immune system of higher animals.
Human Immunodeficiency Virus (HIV) is a member of the lentiviruses, a subfamily of retroviruses. Many retroviruses are well-known carcinogens. HIV per se is not known to cause cancer in humans or other animals, but it does present a formidable challenge to the host. The viral genome contains many regulatory elements which allow the virus to control its rate of replication in both resting and dividing cells. Most importantly, HIV infects and invades cells of the immune system; it breaks down the body""s immune system and renders the patient susceptible to opportunistic infections and neoplasms. The immune defect appears to be progressive and irreversible, with a high mortality rate that approaches 100% over several years.
HIV-1 is trophic and cytopathic for T4 lymphocytes, cells of the immune system which express the cell surface differentiation antigen CD4, also known as OKT4, T4 and leu3. The viral tropism is due to the interactions between the viral envelope glycoprotein, gp120, and the cell-surface CD4 molecules (Dalgleish et al., Nature 312:763-767 (1984)). These interactions not only mediate the infection of susceptible cells by HIV, but are also responsible for the virus-induced fusion of infected and uninfected T cells. This cell fusion results in the formation of giant multinucleated syncytia, cell death, and progressive depletion of CD4 cells in HIV-infected patients. These events result in HIV-induced immunosuppression and its subsequent sequelae, opportunistic infections and neoplasms.
In addition to CD4+ T cells, the host range of HIV includes cells of the mononuclear phagocytic lineage (Dalgleish et al., supra), including blood monocytes, tissue macrophages, Langerhans cells of the skin and dendritic reticulum cells within lymph nodes. HIV is also neurotropic, capable of infecting monocytes and macrophages in the central nervous system causing severe neurologic damage. Macrophage/monocytes are a major reservoir of HIV. They can interact and fuse with CD4-bearing T cells, causing T cell depletion and thus contributing to the pathogenesis of AIDS.
Considerable progress has been made in the development of drugs for HIV-1 therapy during the past few years. There are now 14 drugs approved for use in the U.S., including six nucleoside analog reverse transcriptase inhibitors (AZT, 3TC, ddI, ddC, D4T, and abacavir), three non-nucleoside RT inhibitors (nevirapine, delavirdine, and efavirenz) and five protease inhibitors (saquinavir, ritonavir, indinavir, nelfinavir, and amprenavir). Combinations of these drugs are particularly effective and can reduce levels of viral RNA to undetectable levels in the plasma and slow the development of viral resistance, with resulting improvements in patient health and life span.
Despite these advances, there are still problems with the currently available drug regimens. Many of the drugs exhibit severe toxicities, have other side-effects (e.g., fat redistribution) or require complicated dosing schedules that reduce compliance and thereby limit efficacy. Resistant strains of HIV often appear over extended periods of time even on combination therapy. The high cost of these drugs is also a limitation to their widespread use, especially outside of developed countries.
There is still a major need for the development of additional drugs to circumvent these issues. Ideally these would target different stages in the viral life cycle, adding to the armamentarium for combination therapy, and exhibit minimal toxicity, yet have lower manufacturing costs.
Previously, suksdorfin, i.e., (3xe2x80x2R,4xe2x80x2R)-3xe2x80x2-acetoxy-4xe2x80x2-isovaleryloxy-(+)-cis-khellactone, was isolated as an anti-HIV principle from the fruit of Lomatium suksdorfii. Suksdorfin exhibited inhibitory activity against HIV-1 replication in H9 lymphocyte cells with EC50 value of 1.3 xcexcM, and therapeutic index (TI) value of 140. The discovery of suksdorfin led to the syntheses of khellactone derivatives and led to a second lead compound 3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-(3xe2x80x2R,4xe2x80x2R)-(+)-cis-khellactone (DCK), which showed extremely potent anti-HIV activity with EC50 value of 2.56xc3x9710xe2x88x924 xcexcM and a TI value of 136,719 (Xie, L. et al., J. Med. Chem. 42:2662-2672 (1999)).
Xie, L. et al. describe that alkyl and O-alkyl substituents at the 3-, 4-, and 5-positions of DCK produce derivatives with potent anti-HIV activity (J. Med. Chem. 42:2662-2672 (1999)). DCK derivatives are also described in U.S. Pat. Nos. 5,847,165, 5,637,589, 5,726,204, and 5,612,341.
A need continues to exist for compounds which possess anti-HIV activity with improved biodistribution properties. There is also a need for safe and effective compounds that can be topically applied to vaginal or other mucosa to prevent HIV infection between individuals.
The present invention provides novel 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of Formula I: 
or pharmaceutically acceptable salts, esters, or prodrugs thereof; wherein
R1 and R4 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, xe2x80x94CH2CONH-alkyl, and C1-4 alkyl substituted with one or more of halogen, trifluoromethyl, cyano, hydroxy, amino, monoalkylamino, or dialkylamino, wherein at least one of R1 or R4 is a substituted C1-4 alkyl group;
R2 and R3 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, phenyl, and xe2x80x94CH2CONH-alkyl;
X and Z are independently selected from the group consisting of O, S and NH; and
where the configurations at 3xe2x80x2 and 4xe2x80x2 can be (R) or (S).
The present invention also provides novel 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone compounds of Formula II: 
or pharmaceutically acceptable salts, esters, or prodrugs thereof; wherein
R1 and R4 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, xe2x80x94CH2CONH-alkyl, and C1-4 alkyl substituted with one or more of halogen, trifluoromethyl, cyano, hydroxy, amino, monoalkylamino, or dialkylamino, wherein at least one of R1 or R4 is a substituted C1-4 alkyl group;
R2 and R3 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, phenyl, and xe2x80x94CH2CONH-alkyl; and
where the configurations at 3xe2x80x2 and 4xe2x80x2 can be (R) or (S).
The present invention also provides pharmaceutical compositions, comprising one or more compounds of Formula I, and a pharmaceutically acceptable carrier or diluent. One or more additional pharmaceutically active compounds can also be included in these compositions.
The compounds of Formula I are useful as anti-retroviral agents. Therefore, the present invention provides methods for inhibiting a retroviral infection in cells or tissue of an animal, comprising administering an effective retroviral inhibiting amount of a compound of Formula I. A preferred embodiment provides a method for treating a patient suffering from a retroviral-related pathology, comprising administering to said subject a retroviral inhibiting effective amount of a pharmaceutical composition that includes a compound of Formula I.
The 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of Formula I can be used in a combination therapy with one or more anti-viral agents. Thus, the present invention provides a method of treating a patient suffering from a retroviral-related pathology, comprising administering to said patient a retroviral inhibiting effective amount of a compound of Formula I in combination with one or more anti-viral agents. Preferably, the anti-viral agent is approved for use for HIV-therapy in the U.S.
The present invention also provides a method of preventing transmission of HIV infection between individuals. In particular, the present invention provides a method of preventing transmission of HIV infection from an HIV infected pregnant woman to a fetus, comprising administering to said woman and/or said fetus a retroviral inhibiting effective amount of one or more compounds of Formula I during pregnancy or immediately prior to, at, or subsequent to birth.
Further, the present invention provides a method of preventing transmission of HIV infection during sexual intercourse, comprising applying a retroviral inhibiting effective amount of a topical composition including one or more compounds of Formula I to vaginal or other mucosa prior to sexual intercourse.
Furthermore, the present invention provides a method for making compounds of Formula I.
Additional embodiments and advantages of the invention will be set forth in part in the description as follows, and in part will be obvious from the description, or may be learned by practice of the invention. The embodiments and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of the present invention have the general Formula I: 
or a pharmaceutically acceptable salt, ester, or prodrug thereof; wherein
R1 and R4 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, xe2x80x94CH2CONH-alkyl, and a C1-4 alkyl substituted with one or more of halogen, trifluoromethyl, cyano, hydroxy, amino, monoalkylamino, or dialkylamino, wherein at least one of R1 or R4 is a substituted C1-4 alkyl group;
R2 and R3 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, phenyl, and xe2x80x94CH2CONH-alkyl;
X and Z are independently selected from the group consisting of O, S and NH; and
where the configurations at 3xe2x80x2 and 4xe2x80x2 can be (R) or (S).
Useful compounds included in the general Formula I are 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone compounds of Formula II: 
or pharmaceutically acceptable salts, esters, or prodrugs thereof; wherein
R1 and R4 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, xe2x80x94CH2CONH-alkyl, and C1-4 alkyl substituted with one or more of halogen, trifluoromethyl, cyano, hydroxy, amino, monoalkylamino, or dialkylamino, wherein at least one of R1 or R4 is a substituted C1-4 alkyl group;
R2 and R3 are independently selected from the group consisting of hydrogen, halogen, hydroxy, alkyl, alkoxy, acyloxy, amino, monoalkylamino, dialkylamino, trifluoromethyl, trifluoromethoxy, phenyl, and xe2x80x94CH2CONH-alkyl; and
where the configurations at 3xe2x80x2 and 4xe2x80x2 can be (R) or (S).
The C1-4 alkyl group can be a straight-chain or a branched alkyl group.
Preferred compounds of the present invention are those where R3 and R4 are hydrogen, and R1 is a substituted C1-4 alkyl group. Another group of preferred compounds are those where R1 and R3 are hydrogen, and R4 is a substituted C1-4 alkyl group. Preferably, the C1-4 alkyl group is methyl or ethyl. Preferably, R2 is hydrogen or C1-4 alkyl, preferably methyl. Preferably, R1 and R4 are hydroxymethyl or halomethyl groups or esters thereof.
Suitable substituted alkyl groups include bromomethyl, dibromomethyl, hydroxymethyl, dihydroxymethyl, acetoxymethyl, (dimethylphosphate)methyl, aminomethyl, diethylaminoethyl, and dimethylaminomethyl.
Ester groups are preferably of the type which are relatively readily hydrolyzed under physiological conditions. Examples of pharmaceutically acceptable esters of the compounds of the invention include C1-6 alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C5-7 cycloalkyl esters as well as arylalkyl esters, such as, but not limited to benzyl. C1-4 alkyl esters are preferred. Preferably the esters are selected from the group consisting of alkylcarboxylic acid esters, such as acetic acid esters, and mono- or dialkylphosphate esters, such as methylphoshate ester or dimethylphosphate ester. Esters of the compounds of the present invention can be prepared according to conventional methods.
Preferably, the configurations at 3xe2x80x2 and 4xe2x80x2 are both (R). Also, preferably the O-camphanoyl group is O-(S)-(xe2x88x92)-camphanoyl.
Useful compounds include:
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-bromomethyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-dibromomethyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-acetoxymethyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-hydroxymethyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-bromomethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-acetoxymethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-hydroxymethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-bromomethyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-dibromomethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-bromomethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-acetoxymethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-hydroxymethyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-(dimethylphosphate)methyl-4-methyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-aminomethyl-(+)-cis-khellactone;
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-6-aminomethyl-4-methyl-(+)-cis-khellactone; and
(3xe2x80x2R,4xe2x80x2R)-3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-3-diethylaminomethyl-(+)-cis-khellactone
as well as pharmaceutically acceptable salts and esters thereof.
Useful halo or halogen groups include fluorine, chlorine, bromine and iodine.
Useful alkyl groups include straight-chained and branched C1-10 alkyl groups, more preferably C1-6 alkyl groups, more preferably C1-4 alkyl groups. Typical C1-10 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, 3-pentyl, hexyl and octyl groups.
Useful alkoxy groups include oxygen substituted by one of the C1-10 alkyl groups mentioned above.
Useful acyloxy groups are any C1-6 acyl (alkanoyl) attached to an oxy (xe2x80x94Oxe2x80x94) group, e.g., acetoxy, propionyloxy, butanoyloxy, pentanoyloxy, hexanoyloxy and the like.
An amino group is xe2x80x94NH2.
Useful monoalkylamino and dialkylamino groups are xe2x80x94NHR5 and xe2x80x94NNR5R6, wherein R5 and R6 are C1-10 alkyl groups.
Also, included within the scope of the present invention are the non-toxic pharmaceutically acceptable salts of the compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Examples of pharmaceutically acceptable salts include inorganic and organic acid addition salts such as hydrochloride, hydrobromide, phosphate, sulfate, bisulfate, nitrate, citrate, lactate, tartrate, maleate, fumarate, mandelate, acetate, dichloroacetate, oxalate, valerate, oleate, palmitate, stearate, laureate, borate, benzoate, tosylate, succinate, naphthylate, mesylate, and the like. These may include cations based on the alkali and alkali earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, and the like.
Certain compounds within the scope of Formulae I and II are derivatives referred to as xe2x80x9cprodrugsxe2x80x9d. The expression xe2x80x9cprodrugxe2x80x9d refers to compounds that are rapidly transformed in vivo by an enzymatic or chemical process, to yield the parent compound of the above formulas, for example, by hydrolysis in blood. A thorough discussion is provided by Higuchi, T. and V. Stella in Pro-drugs as Novel Delivery Systems, Vol. 14, A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, American Pharmaceutical Association, Pergamon Press, 1987. Useful prodrugs are esters of compounds of Formulae I and II, wherein at least one of R1 or R4 is a lower alkyl group substituted with one or more hydroxy or halo groups, with a suitable acid. Suitable acids include, e.g., carboxylic acids, sulfonic acids, phosphoric acid or lower alkyl esters thereof, and phosphonic acid or lower alkyl esters thereof. For example, suitable carboxylic acids include alkylcarboxylic acids, such as acetic acid, arylcarboxylic acids and arylalkylcarboxylic acids. Suitable sulfonic acids include alkylsulfonic acids, arylsulfonic acids and arylalkylsulfonic acids. Suitable phosphoric and phosphonic acid esters are methyl or ethyl esters.
Further useful prodrugs of compounds of Formula I and II include those where at least one of R1 or R4 is xe2x80x94(CH2)1-4Y, wherein Y is selected from the group consisting of xe2x80x94Oxe2x80x94C(O)xe2x80x94(CH2)nxe2x80x94CO2H; xe2x80x94Oxe2x80x94C(O)xe2x80x94(CH2)nxe2x80x94NH2; xe2x80x94Oxe2x80x94P(O)(OR)2; and xe2x80x94Oxe2x80x94SO2R, wherein R is xe2x80x94(CH2)nxe2x80x94NH2 or xe2x80x94(CH2)nxe2x80x94CO2H and n is an integer from 1 to 10, preferably from 2 to 6. The free amino and carboxy groups can be converted to pharmaceutically acceptable salts or esters as described above. Prodrugs of the compounds of the present invention can be prepared according to conventional methods.
3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs according to the present invention have been found to possess anti-retroviral, particularly anti-HIV, activity. The analogs of the present invention are expected to have improved water solubility, and enhanced oral bioavailability. Also, due to the improved water solubility, it will be easier to formulate the analogs of the present invention into pharmaceutical preparations. Further, 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs according to the present invention are expected to have improved biodistribution properties.
The invention is also directed to a method for treating a subject infected with HIV-1 by administering at least one of the above-noted 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs, optionally in combination with any one or more of the known anti-AIDS therapeutics or an immunostimulant.
Other features, advantages, embodiments, aspects and objects of the present invention will be clear to those skilled in the areas of relevant art, based upon the description, teaching and guidance presented herein.
The analogs of the present invention have been discovered to have anti-retroviral activity, thus providing suitable compounds and compositions for treating retroviral infections, optionally with additional pharmaceutically active ingredients, such as anti-retroviral, anti-HIV, and/or immuno-stimulating compounds or antiviral antibodies or fragments thereof.
By the term xe2x80x9canti-retroviral activityxe2x80x9d or xe2x80x9canti-HIV activityxe2x80x9d is intended suppression of viral replication whether by any of the following:
(1) viral pro-DNA integration into host cell genome;
(2) retroviral attachment to cells;
(3) viral entry into cells;
(4) cellular metabolism which permits viral replication;
(5) inhibition of intercellular spread of the virus;
(6) synthesis and/or cellular expression of viral antigens;
(7) activity of virus-coded enzymes (such as reverse transcriptase, integrase and proteases); and/or
(8) any known retroviral or HIV pathogenic actions, such as, for example, immunosuppression. Thus, any activity which tends to inhibit any of these mechanisms is xe2x80x9canti-retroviral activityxe2x80x9d or xe2x80x9canti-HIV activity.xe2x80x9d
A 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog of the present invention can be used for treatment of retroviral (e.g., HIV) infection either alone, or in combination with other modes of therapy known in the art. Such modes of therapy can include chemotherapy with drugs, such as, but not limited to, at least one of AZT, ddC, ddA, d4T, ddI, abacavir, nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir or any other antiretroviral drugs or antibodies in combination with each other, or associated with a biologically based therapeutic, such as, for example, soluble CD4, antibodies to CD4, and conjugates of CD4 or anti-CD4, or as additionally presented herein.
Because some of the 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of the present invention are relatively less or substantially non-toxic to normal cells, their utility is not limited to the treatment of established retroviral infections. For example, a 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog according to the present invention can be used in treating blood products, such as those maintained in blood banks. The nation""s blood supply is currently tested for antibodies to HIV. However, the test is still imperfect and samples which yield negative tests can still contain HIV virus. Treating the blood and blood products with the 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of the present invention can add an extra margin of safety by killing any retrovirus that may have gone undetected.
In addition, 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of the present invention can be used as prophylactics to prevent transmission of HIV infection between individuals. For example, the analogs can be administered orally or by injection to an HIV infected pregnant woman and/or fetus during pregnancy or immediately prior to, at, or subsequent to birth, to reduce the probability that the newborn infant becomes infected. Also, the analogs can be administered vaginally immediately prior to childbirth to prevent infection of the infant during passage through the birth canal. Further, the analogs of the present invention can be used during sexual intercourse to prevent transmission of HIV by applying a retroviral inhibiting effective amount of a topical composition including one or more compounds of Formula I or II to vaginal or other mucosa prior to sexual intercourse. For example, the analogs of the present invention can be used to prevent transmission of HIV from an infected male to an uninfected female or vice versa.
Pharmaceutical Compositions
Pharmaceutical compositions of the present invention can comprise at least one of the 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs. Pharmaceutical compositions according to the present invention can also further comprise other anti-viral agents such as, but not limited to, AZT (Glaxo Smith Kline), 3TC (Glaxo Smith Kline), ddI (Bristol-Myers Squibb), ddC (Hoffmann-La Roche), D4T (Bristol-Myers Squibb), abacavir (Glaxo Smith Kline), nevirapine (Boehringher Ingelheim), delavirdine (Pharmacia and Upjohn), efavirenz (DuPont Pharmaceuticals), saquinavir (Hoffmann-La Roche), ritonavir (Abbott Laboratories), indinavir (Merck and Company), nelfinavir (Agouron Pharmaceuticals), amprenavir (Glaxo Smith Kline), adefovir (Gilead Sciences) and hydroxyurea (Bristol-Myers Squibb).
Additional suitable antiviral agents for optimal use with a 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog of the present invention can include, but are not limited to, AL-721 (lipid mixture) manufactured by Ethigen Corporation and Matrix Research Laboratories; Amphotericin B methyl ester; Ampligen (mismatched RNA) developed by DuPont/HEM Research; anti-AIDS antibody (Nisshon Food); 1 AS-101 (heavy metal based immunostimulant); Betaseron (xcex2-interferon) manufactured by Triton Biosciences (Shell Oil); butylated hydroxytoluene; Carrosyn (polymannoacetate); Castanospermine; Contracan (stearic acid derivative); Creme Pharmatex (containing benzalkonium chloride) manufactured by Pharmalec; CS-87 (5-unsubstituted derivative of Zidovudine), Cytovene (ganciclovir) manufactured by Syntex Corporation; dextran sulfate; D-penicillamine (3-mercapto-D-valine) manufactured by Carter-Wallace and Degussa Pharmaceutical; Foscamet (trisodium phosphonoformate) manufactured by Astra AB; fusidic acid manufactured by Leo Lovens; glycyrrhizin (a constituent of licorice root); HPA-23 (ammonium-21-tungsto-9-antimonate) manufactured by Rhone-Poulenc Sante; human immune virus antiviral developed by Porton Products International; Ornidyl (eflornithine) manufactured by Merrell-Dow; nonoxinol; pentamidine isethionate (PENTAM-300) manufactured by Lypho Med; Peptide T (octapeptide sequence) manufactured by Peninsula Laboratories; Phenytoin (Warner-Lambert); Ribavirin; Rifabutin (ansamycin) manufactured by Adria Laboratories; CD4-IgG2 (Progenics Pharmaceuticals) or other CD4-containing or CD4-based molecules; T-20 (Trimeris) or other fusion inhibitors; Trimetrexate manufactured by Warner-Lambert Company; SK-818 (germanium-derived antiviral) manufactured by Sanwa Kagaku; suramin and analogues thereof manufactured by Miles Pharmaceuticals; UA001 manufactured by Ueno Fine Chemicals Industry; and Wellferon (xcex1-interferon) manufactured by Glaxo Smith Kline.
Pharmaceutical compositions of the present invention can also further comprise immunomodulators. Suitable immunomodulators for optional use with a 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog of the present invention in accordance with the present invention can include, but are not limited to: ABPP (Bropririmine); Ampligen (mismatched RNA) DuPont/HEM Research; anti-human interferon-xcex1-antibody (Advance Biotherapy and Concepts); anti-AIDS antibody (Nisshon Food); AS-101 (heavy metal based immunostimulant); ascorbic acid and derivatives thereof; interferon-xcex2; Carrosyn (polymannoacetate); Ciamexon (Boehringer-Mannheim); cyclosporin; cimetidine; CL-246,738 (American Cyanamid); colony stimulating factors, including GM-CSF (Sandoz, Genetics Institute); dinitrochlorobenzene; HE2000 (Hollis-Eden Pharmaceuticals); interferon-xcex1; inteferon-gamma; glucan; hyperimmune gamma-globulin (Bayer); IMREG-1 (leukocyte dialyzate) and IMREG-2 (IMREG Corp.); immuthiol (sodium diethylthiocarbamate) (Institut Merieux); interleukin-1 (Cetus Corporation; Hoffmann-LaRoche; Immunex); interleukin-2 (IL-2) (Chiron Corporation); isoprinosine (inosine pranobex); Krestin (Sankyo); LC-9018 (Yakult); lentinan (Ajinomoto/Yamanouchi); LF-1695 (Fournier); methionine-enkephalin (TNI Pharmaceuticals; Sigma Chemicals); Minophagen C; muramyl tripeptide, MTP-PE (Ciba-Geigy); naltrexone (xe2x80x9cTrexanxe2x80x9d DuPont); Neutropin, RNA immunomodulator (Nippon Shingaku); Remune (Immune Response Corporation); Reticulose (Advanced Viral Research Corporation); shosaikoto and ginseng; thymic humoral factor; TP-05 (Thymopentin, Ortho Pharmaceuticals); Thymosin factor 5 and Thymosin 1; Thymostimulin; TNF (Tumor necrosis factor) manufactured by Genentech; and vitamin B preparations.
The preferred animal subject of the present invention is a mammal. By the term xe2x80x9cmammalxe2x80x9d is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human patients.
The term xe2x80x9ctreatingxe2x80x9d means the administering to subjects a 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone compound for purposes which can include prevention, amelioration, or cure of a retroviral-related pathology.
Medicaments are considered to be provided xe2x80x9cin combinationxe2x80x9d with one another if they are provided to the patient concurrently or if the time between the administration of each medicament is such as to permit an overlap of biological activity.
In one preferred embodiment, at least one 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone compound comprises a single pharmaceutical composition.
Pharmaceutical compositions for administration according to the present invention can comprise at least one 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog according to the present invention in a pharmaceutically acceptable form optionally combined with a pharmaceutically acceptable carrier. These compositions can be administered by any means that achieve their intended purposes. Amounts and regimens for the administration of a 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog according to the present invention can be determined readily by those with ordinary skill in the clinical art of treating a retroviral pathology.
For example, administration can be by parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration can be by the oral route. The dosage administered depends upon the age, health and weight of the recipient, type of previous or concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
Compositions within the scope of this invention include all compositions comprising at least one 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog according to the present invention in an amount effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise about 0.1 to about 100 mg/kg body weight. The preferred dosages comprise about 1 to about 100 mg/kg body weight of the active ingredient. The more preferred dosages comprise about 10 to about 100 mg/kg body weight. The most preferred dosages comprise about 10 to about 50 mg/kg body weight.
Therapeutic administration can also include prior, concurrent, subsequent or adjunctive administration of at least one additional 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analog according to the present invention or other therapeutic agent, such as an anti-viral or immune stimulating agent. In such an approach, the dosage of the second drug can be the same as or different from the dosage of the first therapeutic agent.
Administration of a compound of the present invention can also optionally include previous, concurrent, subsequent or adjunctive therapy using immune system boosters or immunomodulators. In addition to the pharmacologically active compounds, a pharmaceutical composition of the present invention can also contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Preferably, the preparations, particularly those preparations which can be administered orally and which can be used for the preferred type of administration, such as tablets, dragees, and capsules, and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection or orally, contain from about 0.01 to 99 percent, preferably from about 20 to 75 percent of active compound(s), together with the excipient.
Pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients are, e.g., fillers such as saccharide, for example, lactose or sucrose, mannitol or sorbitol; cellulose preparations and/or calcium phosphates, such as tricalcium phosphate or calcium hydrogen phosphate; as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents can be added such as the above-mentioned starches and also carboxymethyl starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl cellulose phthalate are used. Dyestuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
Other pharmaceutical preparations which an be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules which can be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils or liquid paraffin. In addition, stabilizers can be added.
Possible pharmaceutical preparations which can be used rectally include, for example, suppositories which consist of a combination of the active compounds with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the active compounds with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable liphophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions that can contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension can also contain stabilizers.
A pharmaceutical formulation for systemic administration according to the invention can be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulation can be used simultaneously to achieve systemic administration of the active ingredient.
Suitable formulations for oral administration include hard or soft gelatin capsules, dragees, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.
Solid dosage forms in addition to those formulated for oral administration include rectal suppositories.
Prophylactic topical compositions for preventing HIV infection between individuals during childbirth or sexual intercourse include one or more compounds of Formula I or II and at least one pharmaceutically acceptable topical carrier or diluent. The topical composition can be, for example, in the form of an ointment, a cream, a gel, a lotion, a paste, a jelly, a spray, a foam, or a sponge. The dosage amount of a compound of Formula I or II in a prophylactic topical formulation is, in general, less than about 1,000 milligrams, preferably between about 0.01 to about 100 milligrams. The topical formulations can include other prophylactic ingredients. The carrier and diluents should be acceptable in the sense of being compatible with other ingredients of the formulation and not deleterious to the recipient.
Topical prophylactic formulations include those suitable for vaginal, rectal or topical administration. The formulations can, where appropriate, be conveniently presented in discrete dosage units, and can be prepared by any of the methods known in the art of pharmacy. All such methods include the step of bringing the active agent into association with liquid carriers, gels or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.
Prophylactic formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, jelly, foams, or sprays, or aqueous or oily suspensions, solutions or emulsions (liquid formulations) containing suitable carriers known in the art in addition to the active agent. Liquid formulations can contain conventional additives, such as, suspending agents, emulsifying agents, non-aqueous vehicles including edible oils, or preservatives. These formulations are useful to prevent both sexual transmission of HIV and infection of an infant during passage through the birth canal. In one example, the vaginal administration can take place prior to sexual intercourse, or immediately prior to childbirth.
Prophylactic formulations suitable for rectal or vaginal administration having a solid carrier are preferably represented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be formed, for example, mixing one or more compounds of Formula I or II with one or more softened or melted carriers followed by chilling and shaping in molds.
Prophylactic formulations according to the invention can also be in the form of drops formulated with an aqueous or non-aqueous base comprising one or more dispersing agents, solubilizing agents, or suspending agents. Liquid sprays can be delivered from pressurized packs.
Prophylactic formulations according to the invention can be adapted to give sustained delivery. Also, the prophylactic formulations can include other active agents, such as spermicidal agents, antimicrobial agents, and anti-viral agents.
The 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of the present invention can also be administered in the form of an implant when compounded with a biodegradable slow-release carrier. Alternatively, the 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs of the present invention can be formulated as a transdermal patch for continuous release of the active ingredient.
Suitable formulations for topical administration include creams, gels, jellies, mucilages, pastes and ointments. Suitable injectable solutions include intravenous subcutaneous and intramuscular injectable solutions. Alternatively, the 3xe2x80x2,4xe2x80x2-di-O-camphanoyl-(+)-cis-khellactone analogs may be administered in the form of an infusion solution or as a nasal inhalation or spray.
The compounds of the present invention may be prepared using methods known to those skilled in the art. For example, compounds of Formula I where X and Z are both O, i.e., compounds of Formula II, can be prepared as illustrated by exemplary reactions in Schemes 1, 2 and 3. In Schemes 1 and 2, R2 is hydrogen or C1-4 alkyl. In Schemes 1, 2 and 3, R is (S)-camphanoyl. Accordingly, compounds of Formula II can be prepared by brominating 3- or 6-methyl substituted 3xe2x80x2,4xe2x80x2-di-O-(S)-(xe2x88x92)-camphanoyl-(+)-cis-khellactone derivatives with N-bromosuccinimide at a molar ratio of 1:1 to anhydrous benzene to obtain corresponding bromomethyl derivatives (Chilin, A. et al., J. Med. Chem. 42:2936 (1999)). When excess N-bromosuccinimide is used, dibromomethyl derivatives are also obtained. The bromomethyl derivatives can be easily converted to other DCK analogs. The bromomethyl group on the coumarin nucleus can be acetylated with acetic anhydride in the presence of sodium acetate to afford acetoxymethyl derivatives. Subsequently, the acetate ester can be hydrolyzed in EtOH under acidic conditions to afford hydroxymethyl derivatives (Schade, B., et al., J. Org. Chem. 64:9109(1999)). Alternatively, bromomethyl derivatives can be reacted with hexamethylenetetramine followed by hydrolysis in the presence of a catalytic amount of HCl to give aminomethyl derivatives (Doucet, C., et al., J. Med. Chem. 42:4161 (1999)). To prepare dialkylamino derivatives, bromomethyl derivatives can be reacted with, for example, diethylamine in anhydrous toluene to give diethylamino derivatives (Chilin, A., et al., J. Med. Chem. 42:2936 (1999)). Each of these reactions are easy to perform at reflux temperatures. 
The starting compounds in Schemes 1, 2 and 3 can be prepared, for example, as described by Xie, L., et al. (J. Med. Chem. 42:2662-2672 (1999) and J. Med. Chem. 44:664 (2001)). The starting materials for preparing compounds of Formula I can also be prepared by methods described in U.S. Pat. Nos. 5,847,165, 5,637,589, 5,726,204, and 5,612,341.
Compounds of Formula I where X is S can be prepared by methods analogous to those described above.
The starting compounds for preparing compounds of Formula I where X is NH can further be prepared, for example, as described by Yang, Z-Y, et al. (Bioorg. Med. Chem. Lett. 10:1003-1005 (2000)). Compounds of Formula I where X is NH can be prepared, for example, as described in Scheme 4: 
The analogs of Scheme 4 can be deprotected with TFA to obtain the desired compounds of Formula I.
The biological evaluation of HIV-1 inhibition was carried out according to established protocols, (Kashiwada, Y., et al., J. Med. Chem. 39:1016-1017 (1996); Hashimoto, F., et al., Bioorg. and Med. Chem. 5:2133-2143 (1997)). The T cell line, H9, was maintained in continuous culture log-phase growth in complete medium (RPMI 1640 with 10% fetal calf serum supplemented with L-glutamine at 5% CO2 and 37xc2x0 C.). Test samples were first dissolved in dimethyl sulfoxide at a concentration of 10 mg/ml to generate master stocks with dilutions made into tissue culture media to generate working stocks. The following drug concentrations were used routinely for screening: 50, 5, and 0.5 xcexcg/mL. For agents found to be active, additional dilutions were prepared for subsequent testing so that an accurate EC50 value (defined below) could be determined. As the test samples were being prepared, an aliquot of the H9 cell line was infected with HIV-1 (IIIB isolate) while a second aliquot was mock-infected with complete medium. The virus stocks used for these studies typically had a TCID50 value of 1xc3x97106/mL. An amount of virus equal to approximately 6.25xc3x97104 TCID50 was added to the first aliquot of 3.5xc3x97106H9 cells. The second aliquot received tissue culture medium only, and these mock-infected cells were used for toxicity determinations (IC50 defined below). After a 4 h incubation at 37xc2x0 C. and 5% CO2, both cell populations were washed three times with fresh medium and then added to the appropriate wells of a 96-well plate containing various concentrations of the test drug or tissue culture medium (positive infected control/negative drug control). In addition, AZT was assayed during each experiment as a positive drug control. The plates were incubated at 37xc2x0 C. and 5% CO2 for 5 days. Cell-free culture supernatants were collected on Day 5 for use in a p24 antigen-capture ELISA assay to determine virus replication. p24 is the core protein of HIV and therefore is an indirect measure of the amount of virus present in the culture supernatants. Toxicity was determined using a viable dye method (XTT) on mock-infected H9 cells that were treated with test sample, AZT or no drug. If a test sample suppressed viral replication and was toxic to less than 50% of the cells, its effects were reported in the following terms: IC50, the concentration of test sample which was toxic to 50% of the mock-infected H9 cells; EC50, the concentration of the test sample which was able to suppress HIV replication by 50%; and Therapeutic Index (TI), the ratio of IC50 to EC50.
The following examples are illustrative, but not limiting, of the method, compounds and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.